Living microorganisms can be directly examined with the light microscope, but they need fixing and staining to increase visibility, highlight specific morphological features, and preserve them for future study.
The
first step in most bacterial staining procedures is the preparation of a
smear.
Smear
Preparation
Smear
preparation is a process in which bacterial culture is spread uniformly or as a
thin layer on glass slide.
Bacterial Smear
Preparation
The
crucial step in staining is the preparation of a good smear. A good smear
is important to get a good staining result.
Requirements:
•
Culture of Bacteria
•
Glass slides
•
Bunsen burner
•
Inoculating loop
•
Glassware marking pencil/ Permanent Marker
Step I: Preparation of the glass slide:
•
Clean, grease free slides are needed for smear preparation.
•
Grease or oil from the fingers on slides must be removed by washing the slides
with soap and water
•
Finally rinse the slide with 95% alcohol and dry it.
• Hold the slide by their edge.
Step II: Labeling of slides:
•
Proper labelling of the slide is essential- every slide should be labelled
clearly.
•
A lead pencil /permanent CD marker is used to write on the one end of the glass
slide.
Step III: Preparation of smear:
•
An evenly spread smear should be prepared covering area of 15-20mm diameter.
•
Avoid thick and dense smear because thick smear prevent light penetration to
visualize the morphology of cell.
•
A good smear is one that, when dried, appears as a thin whitish layer or film.
The print of textbook should be legible through the smear.
Different
techniques are used for smear preparation depending upon culture media
i. Broth cultures (liquid medium)
•
Re-Suspend the culture by tapping the tube with your finger.
•
Depending on the size of the loop, one or two loopfuls should be applied to the
center of the slide with a sterile inoculating loop and spread evenly over an
area.
•
Allow the smear to air-dry
ii. Culture plates (Solid medium)
•
Suspension is accomplished by spreading the cells in a circular motion in the
drop of water with the loop. This helps to avoid cell clumping.
•
The finished smear should occupy an area about the size of a nickel and should
appear as a translucent, or semitransparent, whitish film
•
Place a drop of water into the circle that has been created on the slide.
•
Using a sterilized and cooled inoculation loop, obtain a very small sample of a
bacterial colony from the culture palte.
•
Gently mix the bacteria into the water drop.
Step IV: Air dry
•
Smear should be allowed to dry completely at room temperature at safe place
Step
V: Fixation of smear:
• The purpose of fixation of smear
is to preserve and prevent smear being washed away during staining.
• Heat fixation - After smear is
air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner
or sprit lamp. Avoid too much heating.
• After heat fix, allow the smear
to cool before staining.
Fixation
The
stained cells seen in a microscope should resemble living cells as closely as
possible.
Fixation
is the process by
which the internal and external structures of cells and microorganisms are
preserved and fixed in position. It inactivates enzymes that might disrupt cell
morphology and toughens cell structures so that they do not change during
staining and observation. A microorganism usually is killed and attached firmly
to the microscope slide during fixation.
There
are two fundamentally different types of fixation.
Heat
fixation is
routinely used to observe procaryotes. Typically, a film of cells (a smear) is
gently heated as a slide is passed through a flame. Heat fixation preserves
overall morphology but not structures within cells.
Chemical
fixation is used
to protect fine cellular substructure and the morphology of delicate microorganisms. Chemical fixatives
penetrate cells and react with cellular components, usually proteins and
lipids, making them inactive, insoluble, and immobile. Common fixative mixtures
contain components such as ethanol, acetic acid, mercuric chloride,
formaldehyde, and glutaraldehyde.
